Microscopic Identification of Microgram Quantities of D - M a n n o s e and D-Lyxose Direct Synthesis of Crystalline D-Mannose and D-Lyxose Phenylhydrazones by Solvent Diffusion Technique LAWRENCE M. WHITE and GERALDINE E. SECOR W e s t e r n Utilization Research Branch, Agricultural Research Service,
3Iicrograni quantities of D-mannose react very rapidly with phenj-lh:-drazine in water to yield a nondescript granular phenylhydrazone. By an extension of the solvent diffusion technique for the identification of D-fructose and L-arabinose, the reaction is controlled to give the characteristic, colorless, crystalline phenylhydrazone with as little as 1 y of pure or 10 to 15 y of chroniatographically separated D-mannose. d sensitive test is given by D-lyxose and a very insensitive one by L-fucose. 3Iany reducing sugars (but not D-mannose), reacted with phen>-lhydrazinein the presence of acetic acid to form a yellow, crystalline, or gellilie phenylosazone. These products aid in the identification of microgram amounts of sugars. The application of the proposed tests to eluates of the D-mannose-D-fructoseL-arabinose area of paper chromatograms is useful in the positive identification of these sugars, which are difficult to separate and identify chromatographically.
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X T H E separation of sugars by paper chromatography, D-mannose travels with or very close t o D-fructose and L-arabinose in most solvents. Spray reagents and sensitive confirmatory tests are available for the positive identification of D-fi-uCtOSc and L-arabinose ( 3 , 4 ) , but neither a spray reagent nor a sensitive confirmatory test has been available for identifying D-mannose in the presence of D-fructose and L-arabinose. This paper presents an extension of the solvent diffusion technique for the identification of D-frLlctOSe ( 4 ) and L-arabinose ( 3 ) t o the microscopic identification of microgram amounts of
U. S. Department
of Agriculture, Albany 7 0, Calif.
n-mannose. Under the same conditions D-lyxose and L-fucose give less sensitive tests. Water is the diffusing solvent and phenylhydrazine is the active reagent for the tests reported here. The application of phenylosazone formation to the identification of microgram quantities of some reducing sugars is also discussed. 4PPARATUS AND REAGENTS
Apparatus for descending paper chromatography. Equipment for eluting sugars from chromatograms and other apparatus as listed previously ( 4 ) . Pure D-mannose, D-lyxose, L-fucose, D-fructose, and L-arabinose. Phenylhydrazine-a.ater reagent. -4dd 1 ml. of water t o 80 mg. of phenylhydrazine, Eastman No. 329, and stir vigorously to promote maximum solution. Stir again immediately before each use. Prepare fresh daily. Phenylhydrazine-acetic acid reagent. Add 1 ml. of 50% (v./v.) acetic acid to 80 mg. of phenylhydrazine and stir until dissolved. Prepare fresh daily. Mineial oil, heavy. Filter paper, Rhatman No. 1 sheets. Ethyl ether, anhydrous, reagent grade. n-Butyl alcohol-ethyl alcohol-\T-ater (10-1-2). Duolite A-4 and Amberlite I R 120-H resins (4). METHOD AND OBSERVATIONS
Preparation of Diffusion Cell. Place an aliquot of a pure sugar solution or a deionized eluate from an ether-washed chromatogram ( 4 )on a microscope slide and allow t o air-dry at room temperature. Place 1 pl. of phenylhydrazine-lvater reagent in the well of the culture slide and assemble and seal the cell with mineral oil ( 4 ) . Observe the progress of solvent and reagent diffusion and the appearance of the reaction product at 25 and lOOX, using both reflected and transmitted llght. Compare with products formed under the same conditions by authentic sugars. R e d e v e 1o p m e n t of Slide under Acidic Conditions. Open the diffusion cell after about Table I. Appearance of Phenj-lhydrazonesand Phenylosazones, Reaction Time, 2 hours, when any o-mannose and Sensitivity of Sugars to Tests present nil1 be nearly comSensitivity, Y pletely p r e c i p i t a t e d a s t h e Time, ChroniatoSugar phenylhydrazone, and quickly Usual Appearance of Reaction Product Hours Pure graphedb Derivative" wipe out any phenylhydrazineD-Mannose phenylColorless, small plates sometimes superimposed, or 0 . 1 t o 2 1 10 water reagent left in the well long, thin, narrow, jagged skeletal blades singly, in hydrazone of the culture slide. Add 1 yl. clusters, or in dendritic patterns of the phenylhydrazine-acetic Fine, straight, long blunt-ended needles in loose clus- 0 . 3 t o 2 5-10 15-25 D-Lyxose phenylacid reagent directly t o the hydrazone ters o r sheaves. Sometimes develops from a gel reaction product or, in the Clusters of very densely packed fine colorless needles 0 . 5 t o 5 50-100 100-200 L-Fucose phenylabsence of such a product, hydrazone in coral or fern pattern often having a sculptured appearance. On aging, long colorless needles may to the solvent pool on the f o r m . Growth confined to well wetted area of slide. slide. Reassemble the cell at M a y develop from a translucent gel once. Kote the insolubility of D-Fructose phenylWell defined yellow-orange small needles in densely 0 . 5 t o 2 3 10 any colorless phenylhydrazone o~acone-1.e.. D packed imperfect spherulitic clusters. Growth present (D-mannose), the disstarts soon as pin points and progresses slowly. 1 I a y glucose phenylosazone form on drier area of slide outside main solvent pool appearance of any colorless D-Lyxose phenylWell defined yellow-orange fine needles in rather per6 t o 16 5 15-25 phenylhydrazone (D-lyxose or osazone fect spherulitic clusters. h l a y also occur as a dense L-fucose), the time required m a t of long needles or filaments. UsuallJ- forms in for a visible yellow phenylosaa pool of solvent zone t o appear, its rate of L-Arabinose phenyl- Light greenish yellow gellike patches. On aging 1 t o 3 5 t o 16 10-15 50 f o r m a t i o n , and appearance days may become semicrystalline in appearance. osazone while forming and after aging. 6 100 y of each of follou-ing pure monosaccharides gave no visible phenylhydrazone after 16 hours' diffusion with C o m p a r e w i t h slides made phenylhydrazine-TT-ater reagent: n-xylose, L-arabinose, D-ribose, L-rhamnose, D-fructose, L-sorbose, D-glucose, and with authentic sugars and D-galactose. All gave a yellow phenylosazone on treatment with phenylhydrazine in acetic acid. b Amount of sugar applied to paper. w i t h s i m i l a r m i x t u r e s of authentic sugars. 1052
1053
V O L U M E 2 8 , N O . 6, J U N E 1 9 5 6 Observations. Table I gives a brief description of the products formed under the conditions of the tests, the approximate time iiyuired for the reaction products to appear, and the approximate amount of sugar required to give a visible product. The times and amounts given in the table are for a single sugar and may be increased by the presence of another sugar RESULTS AND DISCUSSION
Phenylhydrazine is recognized as the reagent of choice for t lie quantitative precipit’ation of D-mannose from aqueous solution
liecause of the almost unique insolubility of the phenylhydrazone :md because other sugars or impurities do not interfere with the precipitation ( 1 ) . These same factors make phenylhydrazine a valuable reagent for the identification of D-mannose on the microgram scale by the solvent diffusion technique. In :iddition to the rather specific nature of this reagent xhen used 3 s directed, it is sufficiently volatile to permit it, as well as the solvent, to diffuse into the sugar; thus, no extraneous crystals appear in the field to offer difficulty in recognizing the reaction product. If phenylhydrazine is applied directly to the sugar Spot, especially in the presence of even a trace of arid, the reaction n-ith D-mannose is so rapid that only a granular, uncharacteristic product is formed and several other sugars will also give a phenylhydrazone or a phenylosazone. However, \\-hen phenylhydrazine is permitted to diffuse slowly from %-ateras described, the ultimate sensitivity of the reaction for o-mannose is not impaired; an easily recognizable, characteristic, crystalline nniannose phenylhydrazone is formed : and interference by the formation of other phenylhydrazones and phenylosazones is greatly reduced. The test for n-mannose is relatively insensitive to interfercnce lrom other sugars, especially D-fructose and L-arabinose, which travel with D-mannose on the paper chromatogram when an irrigating solvent consisting of butyl alcohol, ethyl alcohol, and water is used. Satisfactory k s t s for D-mannose xere obtained when 10 y or more were chromatographed TTith an equal amount of o-fructose and/or Larahinose and r h e n 15 y of n-mannose were chromatographed with 150 y of D-fructose or with 75 -J of L-arabinose. With pure sugars satisfactory tests were obtained for 5 y of o-mannose in the presence of 50 y of o-fructose and for 10 y of D-mannose in the presence of 50 -/ of L-arabinose. However, large amounts of L-arabinose caused an atypical appearance of the reaction product. Twenty micrograms of ot,her pure sugars-D-xylose, Lsorbose, D-glucose, and n-galactose-gave no interference in the test for 10 y of D-mannose. The only other sugars that gave visible reaction products by diffusion of phenylhydrazine from water were D-lyxose and r,-fucose. The phenylhydrazones of these sugars are easily distinguishable from that of D-mannose and from each other by their appearance and by their solubility in over 95% ethyl alcohol or 507, acetic acid. D-Mannose phenylhydrazone remains substantially unchanged when placed over 1 PI. of either of these reagents in the diffusion cell, the D-lyxose product is relatively soluble, and the &fucose product is intermediate in solubility. Table I shows that phenylhydrazine is a sensitive reagent for pure or chromatographically separatrd D-lyxose. However, the presence of other sugars increases the reaction time and the amount of D-lyXOSe required to give a test, or map completely prevent the appearance of the o-lyxose phenylhydrazone. The extent of the inhibition depends on the relative amounts of the sugars. Adequate tests were given by 100 y of pure or chromatographically separated o-lyxose with 25 y of D-xylose, Dribose, or L-fucose, but 50 and 100 y were completely inhibitory. The sensitivity of this reagent for pure and chromatographically stsparated Lfucose is poor. This sugar is better detected by the u s r of 2,4-dinitrophenylhydrnzine (4)or 1,I-diphenylhydrazine (3). If 0-iiiarinose pheriylhydrazonc is allowed to foriii over the
phenj 1hydl.azirie-watrr reagent as characteristic colorless crystals and the slide is then redeveloped by adding acetic acid and more phenylhydrazine directly to the sugar spot, nearly any othei monosaccharide which may be present will form a yellow phenylosazone. The n-mannose phenylhydrazone will remain unchanged or become coated R ith the yellow phenylosazone although the microscopic appearance of sugar phenylosazones synthesized on a semimicro scale at elevated temperature in a neutral, buffeied solution has been used in the identification of sugars (Z), the phenylosazones formed during the redevelopment procedure a t room temperature in the presence of acetic acid do not have a very characteristic appearance. Therefore, the iedevelopment test described above is of substantial value only n hen the number of sugars present is limited, for example, i n the eluate of the D-mannose-D-fructose-L-arabinose area of :i chromatogram. Under the conditions of the redevelopmerit procedure, n-fructose gives a very sensitive test (see Table I ) and Garabinose only a moderately sensitive one. Close obFervation n-hile these phenylosazones are forming and after they have aged permits the identification of D-fructose or Larabinose in the presence of D-mannose. Using the diffusion and redevelopment procedures, adequate tests were obtained for D-mannose and o-fructose when 3 y of pure or 15 y of chromatographically separated D-fructose v a s present with ten times as much D-mannose. The redevelopnient test is too insensitive to detect chromatographically separated L-arabinose in the presence of D-mannose. However, adequate tests for D-mannosr and Larabinose were obtained when 5 , 25, and 50 y of pure D-mannose and 25 y of pure L-arabinose xere present together. The redevelopment test may also be valuable in the analysis of eluates of the D-lyxose-D-xylose-Lfucose-D-ribose area of paper chromatograms when the sugars have not traveled far enough to be quantitativelr separated. During redevelopment the colorless D-lyxose phenylhydrazone formed during the diffusion process dissolves within approximately 0.5 hour and yellow crystalline D-lyxose phenylosazone appears. As shown in Table I, the phenylhydrazone and phenylosazone tests are about equally sensitive for pure or for chromatographically separated D-lyxose Those combinations of D-lyxose with D-xylose, D-ribose, or L-fucose that gave a test for D-lyxose as the phenylhydrazone usually gave a test as the phenylosazone. D-Lyxose that did not form a phenylhydrazone during the diffusion period because of inhibition by any of these three sugars sometimes appeared as the phenylosazone on redevelopment. I n interpreting these results it must be remembered that D-ribose and Lfucose may form gellike phenylosazones under the redevelopment conditions, and that D-xylose, which travels very near to D-lyxose on the chromatogram, does not give a visible phenylhydrazone but does give the same phenylosazone as does D-lyxose because of the structural relations of these sugars. The tests described here and those in the preceding communications ( 3 , 4 ) have been directed primarily toward the identification of D-fructose, tarabinose, and n-mannose in eluates of paper chromatograms. Efforts are being continued to develop solvent diffusion test suitable for the identification of microgram amounts of other difficultly separable sugars and related compounds. LITERATURE CITED
(1) Haar, .i. IV van der, “Anleitung sum Kachweis, zur Trennung und Bestimmung der reinen und aus Glukosiden usw. erhaltenen LIonosaccharide und illdehydsauren,” pp. 231-2, Ge-
bruder Borntraeger, Rerlin, 1920. (2) Hassid, W. Z , LIcCre. dy, R. 31.,IXD.ENG.CHEM.,ANAL.ED 14, 683 (1942) (3) Secor, G. E., White, L. :)I., ANAL.CHEM.27, 1998 (1955). (4) White, L. AI,, Secor, G. E., Ibid., 27, 1016 (1955). R E C E I V E for D review December 1 6 , 1955. Accepted March 20, 1956. The mention of a n y products does not imply t h a t t h e y are endorsed or recommended b y tlie Department of Agriculture over others of a similar nature not nientioned.
